Molecular Mechanisms of Apical Membrane Traffic in Epithelial Cells

上皮细胞顶膜运输的分子机制

• 批准号: 7169917
• 负责人: Rytis Prekeris
• 金额: $ 19.4万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169917
• 关键词: Address; Affinity Chromatography; Apical; Binding; Binding Proteins; Biochemical; Biogenesis; Biological; Biological Assay; Cells; Complex; Data; Defect; Digestive System Disorders; Endosomes; Epithelial; Epithelial Cells; Family; Goals; In Vitro; KRP protein; Kidney; Knowledge; Laboratories; Lead; Lipids; MDCK cell; Mass Spectrum Analysis; Membrane; Membrane Protein Traffic; Membrane Proteins; Microtubules; Molecular; Molecular Motors; Phosphorylation; Phosphorylation Site; Postdoctoral Fellow; Protein Array; Proteins; Publishing; Recruitment Activity; Regulation; Research; Research Personnel; Role; Sorting - Cell Movement; Specificity; Structure; Techniques; Testing; Transport Vesicles; Tubulin; Vesicle; Work; X-Ray Crystallography; Yeasts; analytical ultracentrifugation; apical membrane; base; cell motility; design; domain mapping; in vivo; novel; programs; protein transport; rab GTP-Binding Proteins; research study; trafficking; yeast two hybrid system

DESCRIPTION (provided by applicant): Epithelial cells establish and maintain the polarized distribution of membrane proteins and lipids by continuously sorting them during biogenesis and endocytic cycles. Protein targeting is critical for most epithelial functions. Consequently, its defects are known to lead to a variety of renal and digestive disorders. The main goal of my research is to investigate the mechanisms that regulate Rab 11-dependent apical protein targeting in epithelial cells. Targeting of proteins is achieved through the use of transport vesicles/tubules that fuse with specific target compartments. The vesicle protein, Rab 11, has been shown to be a key molecule involved in apical membrane traffic and may even function as a vesicle "address" tag. In general, Rabs function by recruiting effector proteins to transport vesicles. Recent studies have identified several novel Rab 11-binding proteins, including Rip 11, FIP2, and Eferin/FIP3, also known as FIPs. Based on my published and preliminary data, I hypothesize that (i) Rab 11/Rip11 complexes regulate apical membrane traffic by forming "targeting patches" on transport vesicles, (ii) Rab 11/Rip 11 "targeting patch" is regulated by Rip l 1 phosphorylation, and (iii) Rab 11/Rip 11 complex function by recruiting additional proteins, such as kinesin II to transport vesicles. Four specific aims are designed to further analyze the structure and function of Rab 11/effector complexes. First, the structural basis for Rab 11 interactions with Rip 11 will be investigated using domain mapping and X-ray crystallography. Second, we will use the structural information generated by Aim #1 to determine the role of Rip 11 as well as other FIPs in apical and basolateral targeting using in in vivo transport assays. Third, the role of Rip11 phosphorylation in polarized epithelial traffic will be investigated. Forth, identification and characterization of the proteins interacting with Rab 11/Rip 11 complexes. Characterization of Rab 11/Rip11 complex will provide critical mechanistic information for understanding protein targeting in epithelial cells.

描述（由申请人提供）：上皮细胞通过在生物发生和内吞周期中不断地对膜蛋白和脂质进行分类来建立并维持膜蛋白和脂质的极化分布。蛋白质靶向对于大多数上皮功能至关重要。因此，已知其缺陷会导致多种肾脏和消化系统疾病。我研究的主要目标是研究调节上皮细胞中 Rab 11 依赖性顶端蛋白靶向的机制。蛋白质的靶向是通过使用与特定靶区室融合的运输囊泡/小管来实现的。囊泡蛋白 Rab 11 已被证明是参与顶膜运输的关键分子，甚至可能充当囊泡“地址”标签。一般来说，Rab 通过招募效应蛋白来运输囊泡来发挥作用。最近的研究发现了几种新型 Rab 11 结合蛋白，包括 Rip 11、FIP2 和 Eferin/FIP3（也称为 FIP）。根据我发表的初步数据，我假设 (i) Rab 11/Rip11 复合物通过在运输囊泡上形成“靶向斑块”来调节顶膜运输，(ii) Rab 11/Rip 11“靶向斑块”由 Rip l 1 磷酸化调节，以及 (iii) Rab 11/Rip 11 复合物通过招募其他蛋白质（如驱动蛋白 II）来运输来发挥功能囊泡。设计了四个具体目标来进一步分析 Rab 11/效应器复合物的结构和功能。首先，将使用域图和 X 射线晶体学研究 Rab 11 与 Rip 11 相互作用的结构基础。其次，我们将使用 Aim #1 生成的结构信息来确定 Rip 11 以及其他 FIP 在体内转运测定中的顶端和基底外侧靶向中的作用。第三，将研究 Rip11 磷酸化在极化上皮交通中的作用。第四，与 Rab 11/Rip 11 复合物相互作用的蛋白质的鉴定和表征。 Rab 11/Rip11 复合物的表征将为了解上皮细胞中的蛋白质靶向提供关键的机制信息。